Rational Design of Hydroxylated Thiazole Orange Photocages for Green Light-Triggered DNA Recombination.

The precise control of DNA recombination enables the cell- or time-dependent regulation of gene expression in studies of gene function. Caged estrogen receptor ligands combined with a Cre-ERT2/loxP system are useful tools for light-triggered DNA recombination. However, the photolysis of most caged compounds requires ultraviolet or blue light, which is toxic and displays low tissue penetration. Although a cyanine-based photo-responsive protecting group (PPG) can release estrogen receptor ligands with longer-wavelength light, its low photolytic efficiency requires long illumination times. We developed a caged estrogen receptor ligand with improved green light-responsive PPGs. The rational modification of Hydroxylated Thiazole Orange (HTO) photocages using electron-donating groups (EDGs), such as dimethoxy (DiMeO)-substituted HTO, resulted in high photolytic efficiency (up to ÏµΦ ≈320 M-1  cm-1 ). Theoretical calculations demonstrated that the enhanced photolytic efficiencies were derived from the increased intramolecular charge transfer by EDGs upon excitation. The efficient uncaging of estrogen receptor ligands enabled the control of gene recombination in a ligand-dependent Cre-ERT2/loxP system in live cells.

Heptamethine Cyanine-Based Molecule Release Triggered by Mitochondrial ROS.

Conditionally activated molecule release in live cells would provide spatiotemporal control for the study and intervention of biological processes, e.g., bioactive molecule monitoring and controlled drug release. Mitochondria are the main sites of reactive oxygen species (ROS) production in cells. Here, we report an ROS-triggered molecule release strategy in mitochondria. A molecule IRTO with dual targeting groups was designed by covalently linking IR-780 (a mitochondrial targeted heptamethine cyanine) and 4-aminobutyl-thiazole orange (NH2-TO, a nuclear dye). IRTO diffused into live cells and first accumulated in mitochondria. As the cyanine moiety reacted with mitochondrial ROS directly or with the help of mitochondrial cytochromes, NH2-TO was released, escaped from mitochondria, and finally located in the nucleus. This process could be visualized by fluorescent imaging, i.e., red fluorescence (from the cyanine moiety of IRTO) first located in mitochondria, and green fluorescence (from NH2-TO) appeared and gradually enhanced in the nucleus with the increase of incubation time. The addition of H2O2 or lipopolysaccharide (LPS, an ROS accelerator) could accelerate the release of NH2-TO, whereas N-acetyl-l-cysteine (NAC, an ROS inhibitor) and mitoquinone mesylate (MitoQ, a mitochondrial ROS scavenger) could obviously decrease the release of NH2-TO. These results suggest that IRTO could serve as a fluorescent probe for monitoring ROS in mitochondria and that IR-780 might be a promising endogenous ROS-triggered molecule release platform.

Discriminating young platelets on human leukocyte antigen-I expression highlights their extremely high reactivity potential.

Background: The platelet population is heterogeneous, with different subsets that differ on the basis of their function and reactivity. An intrinsic factor participating in this difference of reactivity could be the platelet age. The lack of relevant tools allowing a formal identification of young platelets prevents so far to draw solid conclusions regarding platelet reactivity. We recently reported that human leukocyte antigen-I (HLA-I) molecules are more expressed on human young platelets. Objectives: The aim of this study was to assess platelet reactivity according to their age based on HLA-I expression level. Methods: Platelet activation was assessed by flow cytometry (FC) for different platelet subsets based on their HLA-I expression. These populations were further cell sorted and their intrinsic properties were determined by FC and electron microscopy (EM). Statistical analyses were performed with GraphPad Prism 5.02 software using two-way ANOVA followed by a Tukey post hoc test. Results: HLA-I expression level allowed the identification of 3 platelet subpopulations regarding to their age (HLA low, dim, and high). HLA-I was reliable to guide platelet cell sorting and highlighted the features of young platelets in the HLA-Ihigh population. In response to different soluble agonists, HLA-Ihigh platelets were the most reactive subset as shown by the level of P-selectin secretion and fibrinogen binding assessed by flow cytometry. Moreover, the highest capacity of HLA-Ihigh platelets to simultaneously express annexin-V and von Willebrand factor or activated αIIbß3 after coactivation with TRAP and CRP indicated that the procoagulant feature of platelets was age-related. Conclusion: The young HLA-Ihigh population is the most reactive and prone to become procoagulant. These results open up new perspectives to investigate deeply the role of young and old platelets.

Metal-Assisted Complexation of Fluorogenic Dyes by Cucurbit[7]uril and Cucurbit[8]uril: A DFT Evaluation of the Key Factors Governing the Host-Guest Recognition.

With the emergence of host-guest systems, a novel branch of complexation chemistry has found wide application in industries such as food, pharmacy, medicine, environmental protection and cosmetics. Along with the extensively studied cyclodextrins and calixarenes, the innovative cucurbiturils (CB) have enjoyed increased popularity among the scientific community as they possess even better qualities as cavitands as compared to the former molecules. Moreover, their complexation abilities could further be enhanced with the assistance of metal cations, which can interestingly exert a dual effect on the complexation process: either by competitively binding to the host entity or cooperatively associating with the CB@guest structures. In our previous work, two metal species (Mg2+ and Ga3+) have been found to bind to CB molecules in the strongest fashion upon the formation of host-guest complexes. The current study focuses on their role in the complex formation with three dye molecules: thiazole orange, neutral red, and thioflavin T. Various key factors influencing the process have been recognized, such as pH and the dielectric constant of the medium, the cavity size of the host, Mn+ charge, and the presence/absence of hydration shell around the metal cation. A well-calibrated DFT methodology, solidly based and validated and presented in the literature experimental data, is applied. The obtained results shed new light on several aspects of the cucurbituril complexation chemistry.

RNA G-quadruplex in live cells lighted-up by a thiazole orange analogue for SCA36 identification.

SCA36 is a neurodegenerative disease mainly caused by the abnormal expansion of the GGGCCT repeat sequence in intron 1 of NOP56. The RNA sequences of this gene are expected to form large amounts of G-quadruplexes in the cytoplasm, which may be a potential intervention and detection target for SCA36. Here, we have developed a small-molecular compound named TCB-1, which shows good selectivity to the G-quadruplex structure, and its fluorescence can be enhanced by hundreds of folds. Interestingly, TCB-1 can avoid lysosome capture, evenly disperse in the cytoplasm, and selectively light up the cytoplasmic RNA G-quadruplexes. This property allows TCB-1 to sensitively detect the increased formation of cytoplasmic RNA G-quadruplexes in SCA36 model cells. This work not only provides new ideas for the design of small-molecule compounds targeting RNA G-quadruplexes in living cells, but also intuitively demonstrates the increased formation of RNA G-quadruplexes caused by NOP56 gene mutation, providing a possible tool for the detection of SCA36.

A Combination of Novel Nucleic Acid Cross-Linking Dye and Recombinase-Aided Amplification for the Rapid Detection of Viable Salmonella in Milk.

Salmonella, as an important foodborne pathogen, can cause various diseases, such as severe enteritis. In recent years, various types of nucleicacid-intercalating dyes have been utilized to detect viable Salmonella. However, in principle, the performance of existing nucleic acid dyes is limited because they depend on the integrity of cell membrane. Herein, based on the metabolic activity of bacteria, a novel DNA dye called thiazole orange monoazide (TOMA) was introduced to block the DNA from dead bacteria. Recombinase-aided amplification (RAA) was then performed to detect viable Salmonella in samples. In this study, the permeability of TOMA to the cell membrane of Salmonella was evaluated via confocal laser scanning microscopy and fluorescence emission spectrometry. The limit of detection (LOD) of the TOMA-RAA method was 2.0 × 104 CFU/mL in pure culture. The feasibility of the TOMA-RAA method in detecting Salmonella was assessed in spiked milk. The LOD for Salmonella was 3.5 × 102 CFU/mL after 3 h of enrichment and 3.5 × 100 CFU/mL after 5 h of enrichment. The proposed TOMA-RAA assay has great potential to be applied to accurately detect and monitor foodborne pathogens in milk and its byproducts.

Ultrafast Excited-State Dynamics of Thiazole Orange.

Thiazole orange (TO), an asymmetric cyanine dye, has been widely used in biomolecular detection and imaging of DNA/ RNA in gels, due to its unique fluorogenic behavior: fluorescence of free dye in aqueous solution is very weak but emission can be significantly enhanced in nucleic-acid-bound dye. Herein we describe the ultrafast excited-state dynamics of free TO in aqueous solution by exploiting both a femtosecond upconversion spectrophotofluorometer and a picosecond time-correlated single-photon counting (TCSPC) apparatus. For the first time, the fluorescence lifetime of TO monomer in water was found to be ∼1 ps, mixed with concurrent solvent relaxation (which was confirmed by the experimental results of TO in DMSO). Even at moderate concentration, this lifetime has an amplitude (a measure of molecular fraction) that significantly dominates other lifetimes, and this is the origin of weak steady state fluorescence of free TO in water. We also found a novel slower decay component around 34 ps. Interestingly and in addition, the lifetime component on the 30-40 ps timescale was also found in TO-γ-Cyclodextrin (CD) complexes. The fraction of this component increased with the addition of γ-CD. Cyclodextrin has been reported to promote the aggregation of TO. Thus, although a very coincidental match of this time constant by one for a torsional process within the cavity can not be ruled out, we ascribe the shared 30-40 ps component to the lifetime of a highly quenched TO dimer experiencing intra-and inter-molecular rearrangement.

A novel photoreactive DNA-binding dye for detecting viable Klebsiella pneumoniae in powdered infant formula.

In addition to Cronobacter spp., Klebsiella pneumoniae is another opportunistic bacterial pathogen present in powdered infant formula (PIF) that can cause pneumonia, septicemia, and other diseases. In this study, a rapid and specific method based on a fluorescence probe was developed for detecting viable K. pneumoniae in PIF samples via the combination of recombinase-aided amplification (RAA) with thiazole orange monoazide (TOMA) dye (the TOMA-RAA assay hereafter). As a novel photosensitive DNA-intercalating dye, TOMA was used to penetrate bacterial cells, including both dead and viable cells, as verified by confocal laser scanning microscopy and fluorescent emission spectrometry. Importantly, the RAA assay exhibited good performance in detecting K. pneumoniae within 40 min at 39°C. Under optimal conditions, the TOMA-RAA assay can detect as low as 2.6 × 103 cfu/mL of K. pneumoniae in pure culture and 2.3 × 104 cfu/g of K. pneumoniae in spiked PIF sample. After 3 h of pre-enrichment, 3 × 100 cfu/g of K. pneumoniae can be detected. Furthermore, the TOMA-RAA assay displayed an excellent anti-interference ability to nontarget bacteria. In short, the proposed method has great potential application for the rapid and accurate detection of viable K. pneumoniae in PIF.

Transport of Molecular Cargo by Interaction with Virus-Like Particle RNA.

Virus-like particles (VLPs) derived from Leviviridae virions contain substantial amounts of cellular and plasmid-derived RNA. This encapsidated polynucleotide serves as a reservoir for the efficient binding of the intercalating dye thiazole orange (TO). Polyethylene glycol (PEG) molecules and oligopeptides of varying length, end-functionalized with TO, were loaded into VLPs up to approximately 50 % of the mass of the capsid protein (hundreds to thousands of cargo molecules per particle, depending on size). The kinetics of TO-PEG binding included a significant entropic cost for the reptation of long chains through the capsid pores. Cargo molecules were released over periods of 20-120 hours following simple reversible first-order kinetics in most cases. These observations define a simple general method for the noncovalent packaging, and subsequent release, of functional molecules inside nucleoprotein nanocages in a manner independent of modifications to the capsid protein.

p-Aminostyryl thiazole orange derivatives for monitoring mitochondrial viscosity in live cells.

Viscosity of cell microenvironment plays a significant role in maintaining the normal life activities of cells. Particularly, the abnormal viscosity in mitochondria is closely associated with lots of diseases and cellular dysfunctions. Herein, we developed a group of p-aminostyryl thiazole orange derivatives with different amino side chains. These probes showed good fluorescence response to viscosity with twisted intramolecular charge transfer mechanism, among them, the probes with diethylamino (TOB), dibutylamino (TOC) and pyrrolidin (TOE) side chains showed better response to the viscosity with 78-fold, 55-fold, and 88-fold fluorescence enhancement in 95% glycerol solution respectively. TOB, TOC, and TOE could enter live cells and mainly located in mitochondria. Treatment HeLa cells with nystatin, lipopolysaccharide or oleic acid caused significant fluorescence enhancement of these probes, suggesting the good potential for monitoring the variation of mitochondrial viscosity, as well as for investigating the related physiological process of inflammation and lipid metabolism.

Rhodamine 6G-Ligand Influencing G-Quadruplex Stability and Topology.

The involvement of G-quadruplex (G4) structures in nucleic acids in various molecular processes in cells such as replication, gene-pausing, the expression of crucial cancer-related genes and DNA damage repair is well known. The compounds targeting G4 usually bind directly to the G4 structure, but some ligands can also facilitate the G4 folding of unfolded G-rich sequences and stabilize them even without the presence of monovalent ions such as sodium or potassium. Interestingly, some G4-ligand complexes can show a clear induced CD signal, a feature which is indirect proof of the ligand interaction. Based on the dichroic spectral profile it is not only possible to confirm the presence of a G4 structure but also to determine its topology. In this study we examine the potential of the commercially available Rhodamine 6G (RhG) as a G4 ligand. RhG tends to convert antiparallel G4 structures to parallel forms in a manner similar to that of Thiazole Orange. Our results confirm the very high selectivity of this ligand to the G4 structure. Moreover, the parallel topology of G4 can be verified unambiguously based on the specific induced CD profile of the G4-RhG complex. This feature has been verified on more than 50 different DNA sequences forming various non-canonical structural motifs.

Near-Infrared Small Molecule as a Specific Fluorescent Probe for Ultrasensitive Recognition of Antiparallel Human Telomere G-Quadruplexes.

In the past 10 years, many fluorescent probes have been developed to recognize G-quadruplexes (G4s) since G4s play an important role in biological systems. However, the selectivity and sensitivity of existing probes for G4s limit their further applications. Herein, we design and synthesize a new probe (TOVJ) by introducing 9-vinyljulolidine into TO. The new probe exhibits almost no fluorescence in an aqueous solution. Upon interacting with G4s, especially the antiparallel G4s, the fluorescence intensity was greatly enhanced (maximum 2742-fold) with a large Stokes shift of 198 nm and the maximum emission peak at 694 nm (near-infrared region). TOVJ showed high sensitivity and selectivity to G4s over other DNA topologies (ssDNA/dsDNA), especially to antiparallel G4s. For antiparallel human telomere G4 detection, the limits of detection of Hum24 and 22AG Na+ were as low as 164 and 231 pM, respectively. This indicates that TOVJ is a highly sensitive fluorescence sensor that can be effectively used for antiparallel human telomere G4 detection. The result of live-cell imaging showed that TOVJ could enter live cells and locate in the mitochondria.

Stacked Thiazole Orange Dyes in DNA Capable of Switching Emissive Behavior in Response to Structural Transitions.

Functional nucleic acids with the capability of generating fluorescence in response to hybridization events, microenvironment or structural changes are valuable as structural probes and chemical sensors. We now demonstrate the enzyme-assisted preparation of nucleic acids possessing multiple thiazole orange (TO) dyes and their fluorescent behavior, that show a spectral change from the typical monomer emission to the excimer-type red-shifted emission. We found that the fluorescent response and emission wavelength of the TO dyes were dependent on both the state of the DNA structure (single- or double-stranded DNA) and the arrangement of the TO dyes. We showed that the fluorescent behavior of the TO dyes can be applied for the detection of RNA molecules, suggesting that our approach for preparing the fluorescent nucleic acids functionalized with multiple TO dyes could be useful to design a fluorescence bioimaging and detection technique of biomolecules.

Broad Applications of Thiazole Orange in Fluorescent Sensing of Biomolecules and Ions.

Fluorescent sensing of biomolecules has served as a revolutionary tool for studying and better understanding various biological systems. Therefore, it has become increasingly important to identify fluorescent building blocks that can be easily converted into sensing probes, which can detect specific targets with increasing sensitivity and accuracy. Over the past 30 years, thiazole orange (TO) has garnered great attention due to its low fluorescence background signal and remarkable 'turn-on' fluorescence response, being controlled only by its intramolecular torsional movement. These features have led to the development of numerous molecular probes that apply TO in order to sense a variety of biomolecules and metal ions. Here, we highlight the tremendous progress made in the field of TO-based sensors and demonstrate the different strategies that have enabled TO to evolve into a versatile dye for monitoring a collection of biomolecules.

A mitochondria-targeted thiazoleorange-based photothermal agent for enhanced photothermal therapy for tumors.

Organic small molecules with near-infrared (NIR) absorption hold great promise as the phototheranostic agents for clinical translation by virtue of their inherent merits such as well-defined chemical structure, high purity and good reproducibility. Probes that happen to be based on cyanine dyes exhibit strong NIR-absorbing and efficient photothermal conversion, representing a new class of photothermal agents (PAs) for photothermal therapy (PTT), and taking into account the heat susceptibility of Mitochondria (Mito), we designed and prepared a mitochondria-targeted organic small molecule (Mito-BWQ) based on thiazole orange maternal unit that can effectively kill tumor cells through the hyperpyrexia generated in the lesions under exogenous laser irradiation. The Confocal laser scanning microscope was employed to determine the preferential targeting of Mito-BWQ to the mitochondria of MCF-7 cells and U87 cells. When subjected to 600 nm laser radiation, Mito-BWQ produced an increase in temperature in test systems and this increase was dependent on both the laser power and probe concentration. In vitro tests, cytotoxicity was observed when cells were incubated with Mito-BWQ and exposed to laser irradiation. The PTT in vivo also showed that Mito-BWQ performed remarkably in tumor inhibition. This study thus provides a vital starting point for the creation of thiazole orange-based PTT formulations and promotes further advances in the field of PAs-based anticancer research and therapy.

DNA/RNA Fluorescence Imaging by Synthetic Nucleic Acids.

Fluorescence imaging of nucleic acids is a key technology needed to understand gene expression and the resulting changes in cellular function. This chapter focuses on sequence-specific fluorescence imaging of nucleic acids in cells using fluorescent nucleic acids. The design and preparation of fluorescent nucleic acids and their application to fluorescence imaging of intracellular nucleic acids are introduced.

Photophysics of thiazole orange in deep eutectic solvents.

Photophysics and torsional dynamics of thiazole orange (TO) as a function of temperature have been studied in two deep eutectic solvents (DESs) using spectroscopic techniques. Two DESs are used as a solvent namely DES-I (choline chloride + urea, mole ratio 1: 2) and DES-II (N,N diethyl ethanol ammonium chloride + urea, mole ratio 1: 2). We explore the influence of DESs on the photophysical properties of TO. The fluorescence quantum yield and fluorescence lifetime of TO decreases with increasing temperature due to thermal deactivation. At higher temperature, fluorescence quantum yield of TO decreases in DESs may be due to the molecular rotor nature of TO, with the benzothiazole and quinoline ring of this dye being able to be rotated relative to each other in the excited state. In these solvents, the free volume idea was found to provide a truthful report of the solvent viscosity-temperature behavior, and the probe torsional dynamics. Fluorescence lifetime imaging microscopy (FLIM) was used to insight and observed the distribution of lifetime of TO in the surface of both DESs. The contact angle was determined to show the hygroscopic nature of the DESs.

Comparison of Two DNA Aptamers for Dopamine Using Homogeneous Binding Assays.

Dopamine is an essential neurotransmitter and its detection is important for bioanalytical chemistry. Two very different DNA aptamers have been reported for dopamine, one derived from an RNA aptamer (named Apt1) and other obtained via direct aptamer selection (named Apt2). In this study, we used four homogeneous binding assays to compare these two DNA dopamine aptamers. Thiazole orange (TO) fluorescence assay indicated that the Apt2 specifically bound with dopamine with a Kd of 2.37 µM, which was consistent with that from the isothermal titration calorimetry (ITC) assay. However, Apt1 had much less TO fluorescence change and also no signal from ITC. By labeling the two ends of the two aptamers by a fluorophore and a quencher, the aptamer beacons showed binding of dopamine only for Apt2. Finally, the label-free AuNP-based colorimetric assay showed no difference between these two aptamer sequences, and even non-binding random DNA showed the same response, indicating that AuNPs were not a good probe for detecting dopamine. According to the data, Apt1 does not appear to be able to bind dopamine specifically, while Apt2 showed specific binding and could be used for developing related biosensors.

A colorimetric and fluorometric based dual readout approach for effective heparin sensing.

Devising fluorescence-based turn-on probes for the specific and sensitive detection of Heparin is of utmost clinical importance. In this contribution, we have identified a molecular rotor based asymmetric cyanine probe, thiazole orange (TO), which enables an efficient colorimetric and fluorimetric detection of Heparin. TO undergoes the formation of emissive H-aggregates upon interaction with Heparin that display an impressive emission enhancement of ~22 fold together with drastic changes in the absorption spectra that yields a prominent colour change in the solution from orange to yellow. These seldom reported emissive H-aggregates of TO, serve as an efficient platform for Heparin detection with a LOD of 19 nM, fluorometrically and 34 nM, colorimetrically. The TO-Heparin complex is also accompanied by a large change in the excited-state lifetime. The TO-Heparin complex has been further utilized for the detection of Protamine, which is the only medically affirmed antitoxin of Heparin. Overall, our sensing system offers several advantages, such as, simple, dual read-out, economic and specific detection of Heparin with longer excitation and emission wavelength, rapid naked eye detection and utilizes an in-expensive commercially available fluoprophore, TO. Most importantly, our sensing system also displays a good performance in the biologically complex human serum matrix.

Synthesis of fluorescent G-quadruplex DNA binding ligands for the comparison of terminal group effects in molecular interaction: Phenol versus methoxybenzene.

A number of new fluorescent nucleic acid binding ligands were synthesized by utilizing the non-specific thiazole orange dye as the basic scaffold for molecular design. Under simple synthetic conditions, the molecular scaffold of thiazole orange bridged with a terminal side-group (phenol or methoxybenzene) becomes more flexible because the newly added ethylene bridge is relatively less rigid than the methylene of thiazole orange. It was found that these molecules showed better selectivity towards G-quadruplex DNA structure in molecular interactions with different type of nucleic acids. The difference in terms of induced DNA-ligand interaction signal, selectivity, and binding affinity of the ligands with the representative nucleic acids including single-stranded DNA, double-stranded DNA, telomere and promoter G4-DNA and ribosomal RNA were investigated. The position of the terminal methoxyl groups was found showing strong influence both on binding affinity and fluorescent discrimination among 19 nucleic acids tested. The ligand with a methoxyl group substituted at the meta-position of the styryl moiety exhibited the best fluorescent recognition performance towards telo21 G4-DNA. A good linear relationship between the induced fluorescent binding signal and the concentration of telo21 was obtained. The comparison of ligand-DNA interaction properties including equilibrium binding constants, molecular docking, G4-conformation change and stabilization ability for G4-structures was also conducted. Two cancer cell lines (human prostate cancer cell (PC3) and human hepatoma cell (hepG2)) were selected to explore the inhibitory effect of the ligands on the cancer cell growth. The IC50 values obtained in the MTT assay for the two cancer cells were found in the range of 3.4-10.8 µM.